Method of treating diarrhea in a canine

ABSTRACT

According to the invention there is provided a strain of lactic acid bacteria of the species  Bifidobacteria pseudolongum  obtainable by isolation from resected and washed canine gastrointestinal tract having a probiotic activity in animals. Methods of use and compositions comprising the  Bifidobacteria pseudolongum  of the present invention are also provided.

CROSS REFERENCE TO RELATED APPLICATION

This Application is a continuation of U.S. application Ser. No.11/013,117, filed on Dec. 15, 2004 now abandoned, which claims thebenefit of and priority to U.S. Provisional Application No. 60/531,099,filed on Dec. 19, 2003.

FIELD OF THE INVENTION

The present invention relates to the field of probiotic micro-organisms,more specifically canine probiotic lactic acid bacteria and methods ofuse.

BACKGROUND OF THE INVENTION

The defense mechanisms to protect the mammalian gastrointestinal (GI)tract from colonisation by pathogenic bacteria are highly complex. TheGI tract of most mammals are colonised by native microflora, andinvasive pathogenic micro-organisms. In a healthy individual, thesecompeting microflora are in a state of equilibrium. Modification of theintestinal microflora equilibrium may lead to or prevent many GIdisorders, both in humans, and other mammalian species, such ascompanion animals including cats, dogs and rabbits. The well being ofcompanion animals is closely related to their feeding and GI health, andmaintenance of the intestinal microflora equilibrium in these animalsmay result in healthier pets.

The number and composition of the intestinal microflora tend to bestable, although age and diet may modify it. Gastric acidity, bile,intestinal peristalsis and local immunity are factors thought to beimportant in the regulation of bacterial flora in the small intestine ofhuman beings and various other mammals. Often pet GI disorders,including those found in canines and felines, are linked to bacterialovergrowth and the production of enterotoxins by pathogenic bacteria.These factors disrupt the intestinal microflora equilibrium and canpromote inflammation and aberrant immune responses.

During the last few years, research has begun to highlight some valuablestrains of bacteria and their potential use as probiotic agents.Probiotics are considered to be preparations of bacteria, either viableor dead, their constituents such as proteins or carbohydrates, orpurified fractions of bacterial ferments that promote mammalian healthby preserving and promoting the natural microflora in the GI tract, andreinforcing the normal controls on aberrant immune responses. It isbelieved by some that probiotic bacteria are more effective when derivedfrom the species, or a closely related species to the individualintended to be treated. Therefore, there is a need for probiotic strainsderived from companion animals to be used for companion animals, thatare different to those derived from humans.

WO 01/90311 discloses probiotic micro-organisms isolated from faecalsamples obtained from cats and dogs having probiotic activity. However,these bacteria were obtained from faecal samples, and may not form partof the natural intestinal microflora present in the upper portion of theGI tract.

Consequently, there is a need to provide strains of bacteria obtainableby isolation from the natural intestinal microflora present in the upperportion of the GI tract that are particularly adapted for companionanimals, and have been selected for their probiotic properties andability to survive processing, and to incorporate these strains intocompositions that are suitable for their use.

SUMMARY OF THE INVENTION

According to the invention there is provided a strain of lactic acidbacteria of the species Bifidobacteria pseudolongum obtainable byisolation from resected and washed canine gastrointestinal tract havinga probiotic activity in animals.

In a preferred embodiment, the lactic acid bacterial strain is aBifidobacteria pseudolongum having a 16s-23s spacer region DNA sequencehaving at least 93% homology to SEQ. ID NO. 1.

In a further preferred embodiment, the lactic acid bacterial strain isBifidobacteria pseudolongum AHC7 (NCIMB 41199).

Furthermore, the present invention is directed towards providing uses ofBifidobacteria pseudolongum bacteria obtainable by isolation fromresected and washed canine gastrointestinal tract for maintaining andimproving pet health, and compositions comprising the lactic acidbacteria.

These and other features, aspects, and advantages of the presentinvention will become evident to those skilled in the art from a readingof the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates the inhibition of the growth in vitro of Salmonellatyphimurium by the Bifidobacteria pseudolongum bacteria of the presentinvention according to methodology set out in example 2.

FIG. 2 demonstrates the inhibition of the growth in vitro of Listeriamonocytogenes by the Bifidobacteria pseudolongum bacteria of the presentinvention according to methodology set out in example 2.

FIG. 3 demonstrates the inhibition of the growth in vitro of Listeriainnocua by the Bifidobacteria pseudolongum bacteria of the presentinvention according to methodology set out in example 2.

FIG. 4 demonstrates the inhibition of the growth in vitro of Escherichiacoli 0157H45 by the Bifidobacteria pseudolongum bacteria of the presentinvention according to methodology set out in example 2.

FIG. 5 demonstrates the in vitro acid stability of the Bifidobacteriapseudolongum bacteria of the present invention according to methodologyset out in example 3.

FIG. 6 demonstrates the growth characteristics of the Bifidobacteriapseudolongum bacteria of the present invention in the presence of 0.5%,1% and 5% porcine bile salts.

FIG. 7 demonstrates the in vitro ability of the Bifidobacteriapseudolongum bacteria of the present invention to adhere to HT-29 gutepithelial cells.

DETAILED DESCRIPTION OF THE INVENTION

This application contains a sequence listing, described hereinafter,which is represented by a paper copy of the sequence listing attached tothe amendment of Aug. 1, 2008, which is hereby incorporated by referenceherein.

Sequences

SEQ. ID NO. 1—16s-23s intergenic spacer nucleotide sequence fromBifidobacteria pseudolongum AHC7 (NCIMB 41199).

SEQ. ID NO. 2—Primer sequences for 16s-23s DNA sequence analysis.

Bacterial Deposit Numbers

The table below indicates Bifidobacteria pseudolongum strains that areexamples of the present invention. The bacterial strains are depositedwith the National Collections of Industrial Food and Marine Bacteria,Ltd. (NCIMB, Ltd.), Ferguson Building, Craibstone Estate, Bucksburn,Aberdeen AB21 9YA, Scotland, UK, under deposit number NCIMB 41199, onSep. 4, 2003.

16 s-23s Strain Deposit Number Sequence Bifidobacteria pseudolongum AHC7NCIMB 41199 SEQ. ID NO. 1

All weights, measurements and concentrations herein are measured at 25°C. on the composition in its entirety, unless otherwise specified.

Unless otherwise indicated, all percentages of compositions referred toherein are weight percentages and all ratios are weight ratios.

Unless otherwise indicated, all molecular weights are weight averagemolecular weights.

Unless otherwise indicated, the content of all literature sourcesreferred to within this text are incorporated herein in full byreference.

Except where specific examples of actual measured values are presented,numerical values referred to herein should be considered to be qualifiedby the word “about”.

Within the following description, the abbreviation CFU (“colony-formingunit”) designates the number of bacterial cells revealed bymicrobiological counts on agar plates, as will be commonly understood inthe art.

As used herein, the term “mutants thereof” includes derived bacterialstrains having at least 93% homology, preferably at least 96% homology,more preferably 98% homology to the 16s-23s intergenic spacerpolynulceotide sequence of a referenced strain, but otherwise comprisingDNA mutations in other DNA sequences in the bacterial genome.

As used herein, the term “DNA mutations” includes natural or inducedmutations comprising at least single base alterations includingdeletions, insertions, transversions, and other DNA modifications knownto those skilled in the art, including genetic modification introducedinto a parent nucleotide or amino acid sequence whilst maintaining atleast 50% homology to the parent sequence. Preferably, the sequencecomprising the DNA mutation or mutations has at least 60%, morepreferably at least 75%, more preferably still 85% homology with theparental sequence. As used herein, sequence “homology” can be determinedusing standard techniques known to those skilled in the art. Forexample, homology may be determined using the on-line homology algorithm“BLAST” program, publicly available athttp://www.ncbi.nlm.nih.gov/BLAST/.

As used herein “genetic modification” includes the introduction ofexogenous and/or endogenous DNA sequences into the genome of an organismeither by insertion into the genome of said organism or by vectorsincluding plasmid DNA or bacteriophage as known by one skilled in theart, said DNA sequence being at least two deoxyribonucleic acid bases inlength.

As used herein, “companion animal” means a domestic animal. Preferably,“companion animal” means a domestic canine, feline, rabbit, ferret,horse, cow, or the like. More preferably, “companion animal” means adomestic canine or feline.

Bifidobacteria Pseudolongum Strains

The first aspect of the present invention comprises a strain ofBifidobacteria pseudolongum obtainable by isolation from resected andwashed canine gastrointestinal tract having probiotic activity inanimals. Probiotics are micro-organisms, either viable or dead,processed compositions of micro-organisms, their constituents such asproteins or carbohydrates, or purified fractions of bacterial fermentsthat beneficially affect a host. The general use of probiotic bacteriais in the form of viable cells. However, it can be extended tonon-viable cells such as killed cultures or compositions containingbeneficial factors expressed by the probiotic bacteria. This may includethermally killed micro-organisms, or micro-organisms killed by exposureto altered pH or subjected to pressure. For the purpose of the presentinvention, “probiotics” is further intended to include the metabolitesgenerated by the micro-organisms of the present invention duringfermentation, if they are not separately indicated. These metabolitesmay be released to the medium of fermentation, or they may be storedwithin the micro-organism. As used herein “probiotic” also includesbacteria, bacterial homogenates, bacterial proteins, bacterial extracts,bacterial ferment supernatants, and mixtures thereof, which performbeneficial functions to the host animal when given at a therapeuticdose.

It has been found that strains of Bifidobacteria pseudolongum obtainableby isolation directly from resected and washed GI tract of mammals areadherent to the GI tract following feeding of viable bacterial cells,and are also significantly immunomodulatory when fed to animals inviable, non-viable or fractionated form. Without being bound by theory,it is believed that the Bifidobacteria pseudolongum obtainable byisolation from resected and washed GI tract closely associate with thegut mucosal tissues. Without further being bound by theory, this isbelieved to result in the probiotic Bifidobacteria pseudolongum of thepresent invention generating alternative host responses that result inits probiotic action. It has been found that probiotic bacteriaobtainable by isolation from resected and washed GI tract can modulatethe host's immune system via direct interaction with the mucosalepithelium, and the host's immune cells. This immunomodulation, inconjunction with the traditional mechanism of action associated withprobiotic bacteria, i.e. the prevention of pathogen adherence to the gutby occlusion and competition for nutrients, results in theBifidobacteria pseudolongum of the present invention being highlyefficacious as a probiotic organism.

The Bifidobacteria pseudolongum of the present invention, obtainable byisolation from resected and washed canine GI tract, have in vitroanti-microbial activity against a number of pathogenic bacterialstrains/species, as measured by zones of inhibition or bacterial growthinhibition assays known to those skilled in the art. Without being boundby theory, it is believed that this in vitro anti-microbial activity isindicative of potential probiotic activity in vivo in animals,preferably companion animals such as canines and felines. The lacticacid bacteria of the present invention preferably have in vitroanti-microbial activity against Salmonella typhimurium, Listeriamonocytogenes, Listeria innocua or Eschericia coli, more preferably amixture of these strains, more preferably still, all of these strains.

Without being bound by theory, it is believed that the anti-microbialactivity of the Bifidobacteria pseudolongum bacteria of the presentinvention may be the result of a number of different actions by theBifidobacteria pseudolongum bacteria herein. It has previously beensuggested in the art that several strains of bacteria isolated fromfaecal samples exert their probiotic effect in the GI tract followingoral consumption by preventing the attachment of pathogenic organisms tothe gut mucosa by occlusion. This requires oral consumption of “live” orviable bacterial cells in order for a colony of bacteria to beestablished in the gut. However, it is believed that the Bifidobacteriapseudolongum of the present invention, obtainable by isolation fromresected and washed canine GI tract, whilst exerting some probioticeffect due to occlusion if given in a viable form, may deliver asubstantial probiotic effect in either the viable or non-viable form dueto the production during fermentation in vitro of a substance orsubstances that either inhibit the growth of or kill pathogenicmicro-organisms, and/or alter the host animal's immune competence. Thisform of probiotic activity is desirable, as the bacteria of the presentinvention can be given as either viable or non-viable cultures orpurified fermentation products and still deliver a beneficialtherapeutic effect to the host animal.

Preferably, the Bifidobacteria pseudolongum bacteria of the presentinvention are able to maintain viability following transit through theGI tract. This is desirable in order for live cultures of the bacteriato be taken orally, and for colonisation to occur in the intestines andbowel following transit through the oesophagus and stomach. Colonisationof the intestine and bowel by the lactic acid bacteria of the presentinvention is desirable for long-term probiotic benefits to be deliveredto the host. Oral dosing of non-viable cells or purified isolatesthereof induces temporary benefits, but as the bacteria are not viable,they are not able to grow, and continuously deliver a probiotic effectin situ. As a result this may require the host to be dosed regularly inorder to maintain the health benefits. In contrast, viable cells thatare able to survive gastric transit in the viable form, and subsequentlycolonise by adhering to and proliferating on the gut mucosa are able todeliver probiotic effects continuously in situ.

Therefore, it is preferable that the lactic acid bacteria of the presentinvention maintain viability after suspension in a media having a pH of2.5 for 1 hour. As used herein, “maintain viability” means that at least25% of the bacteria initially suspended in the test media are viableusing the plate count method known to those skilled in the art.Preferably, “maintain viability” means that at least 50% of the bacteriainitially suspended are viable. It is desirable for the lactic acidbacteria of the present invention to maintain viability followingexposure to low pH as this mimics the exposure to gastric juices in thestomach and upper intestine in vivo following oral consumption inanimals.

Furthermore, it is preferable that the lactic acid bacteria of thepresent invention have a growth of at least 33% when in the presence ofat least 0.5% porcine bile salts. Growth, as used herein is described infurther detail in example 3. More preferably, the bacteria of thepresent invention have a growth of at least 33% when in the presence ofat least 1% porcine bile salts. Without being bound by theory it isbelieved that the lactic acid bacteria of the present invention, capableof growth in the presence of at least 0.5% porcine bile salts, are ableto survive the conditions present in the intestine. This is thought tobe a result of the addition of porcine bile to the culture mediummimicking the conditions of the intestine.

Further still, it is preferable that the Bifidobacteria pseudolongumbacteria of the present invention have significant adhesion to gutepithelial cells in vitro. As used herein, “significant adhesion” meansat least 4% of the total number of lactic acid bacteria co-incubatedwith the epithelial cells in vitro adhere to the epithelial cells. Morepreferably, at least 6% of bacterial cells co-incubated adhere toepithelial cells in vitro. Without being bound by theory, it is believedthat gut epithelial cell adherence in vitro is indicative of the lacticacid bacteria's ability to colonise the GI tract of an animal in vivo.

The 16s-23s intergenic polynucelotide sequence is known to those skilledin the art as the sequence of DNA in the bacterial genome that can beused in order to identify different species and strains of bacteria.This intergenic polynucelotide sequence can be determined by the methoddetailed below in example 4.

In a preferred embodiment of the present invention, the strain ofBifidobacteria pseudolongum has a 16s-23s intergenic polynucleotidesequence that has at least 93%, preferably at least 96%, more preferablyat least 99% homology with the polynucleotide sequence according to SEQ.ID NO. 1. More preferably, the strain of lactic acid bacteria accordingto the present invention has a 16s-23s polynucelotide sequence accordingto SEQ. ID NO. 1. More preferably still, the strain of lactic acidbacteria according to the present invention is Bifidobacteriapseudolongum strain NCIMB 41199 (AHC7), or a mutant thereof.

The strain of lactic acid bacteria of the genus Bifidobacteriapseudolongum obtainable by isolation from resected and washed caninegastrointestinal tract can be used to deliver probiotic benefitfollowing oral consumption in animals, preferably companion animals orhumans. This probiotic benefit generally maintains and improves theoverall health of the animal. Non-limiting elements of animal health andphysiology that benefit, either in therapeutically relieving thesymptoms of, or disease prevention by prophylaxis include inflammatorydisorders, immunodeficiency, inflammatory bowel disease, irritable bowelsyndrome, cancer (particularly those of the gastrointestinal and immunesystems), diarrhoeal disease, antibiotic associated diarrhoea,appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer'sdisease, amyloidosis, rheumatoid arthritis, arthritis, joint mobility,diabetes mellitus, insulin resistance, bacterial infections, viralinfections, fungal infections, periodontal disease, urogenital disease,surgical associated trauma, surgical-induced metastatic disease, sepsis,weight loss, weight gain, excessive adipose tissue accumulation,anorexia, fever control, cachexia, wound healing, ulcers, gut barrierinfection, allergy, asthma, respiratory disorders, circulatorydisorders, coronary heart disease, anaemia, disorders of the bloodcoagulation system, renal disease, disorders of the central nervoussystem, hepatic disease, ischaemia, nutritional disorders, osteoporosis,endocrine disorders, and epidermal disorders. Preferred are treatment ofthe gastrointestinal tract, including treatment or prevention ofdiarrhoea; immune system regulation, preferably the treatment orprevention of autoimmune disease and inflammation; maintaining orimproving the health of the skin and/or coat system, preferably treatingor preventing atopic disease of the skin; ameliorating or reducing theeffects of aging, including mental awareness and activity levels; andpreventing weight loss during and following infection.

The treatment of the disorders disclosed above may be measured usingtechniques known to those skilled in the art. For example, inflammatorydisorders including autoimmune disease and inflammation may be detectedand monitored using in vivo immune function tests such as lymphocyteblastogenesis, natural killer cell activity, antibody response tovaccines, delayed-type hypersensitivity, and mixtures thereof. Suchmethods are briefly described herein, but well known to those skilled inthe art.

-   -   1. Lymphocyte blastogenesis: This assay measures the        proliferative response in vitro of lymphocytes isolated from        fresh whole blood of test and control animals to various        mitogens and is a measure of overall T- and B-cell function.        Briefly, peripheral blood mononucleocytes (PBMC) are isolated        from whole blood by Ficoll-Hypaque density centrifugation        methods known to those skilled in the art. The isolated PBMCs        are washed twice in RPMI 1640 cell media supplemented with        HEPES, L-glutamine and penicillin/streptomycin. The washed cells        are resuspended in RPMI 1640, counted, and the cell density        adjusted appropriately. The 2'10⁵ cells are exposed to a range        of concentrations (0.1 □g/ml to 100 □g/ml) of various mitogens,        some examples of which include pokeweed mitogen (Gibco),        phytohaemagglutinin (Gibco) and conconavalin A (Sigma) in        triplicate for 72 hours at 37° C. and 5% CO₂ with 10% foetal        bovine serum (Sigma). At 54 hours the cells are pulsed with 1        □Ci ³H-thymidine, and the cells harvested and scintillation        counts read on a TopCount NXT at 72 hours.    -   2. Natural killer cell activity: As described in U.S. Pat. No.        6,310,090, this assay measures the in vitro effector activity of        natural killer cells isolated from fresh whole blood of test and        control animals. Natural killer cells are a component of the        innate immune function of a mammal. Canine thyroid        adenocarcinoma cells were used as target cells in assessing NK        cell cytotoxic activity. This cell line was previously shown to        be susceptible to killing by canine NK cell. Target cells were        cultured in a T75 flask with 20 mL minimum essential medium        (MEM; Sigma Chem. Co., St. Louis, Mo.) supplemented with 10%        fetal calf serum (FCS), 100 U/mL of penicillin and 100 □g/mL of        streptomycin. When confluent, target cells were trypsinized,        washed 3 times and resuspended to 5×10⁵ cells/mL in complete        medium (RPMI-1640+10% FCS+100 U/mL of penicillin+100 □g/mL of        streptomycin). Triplicate 100 .□L aliquots of the target cells        were pipetted into 96-well U-bottom plates (Costar, Cambridge,        Mass.) and incubated for 8 hours to allow cell adherence.        Lymphocytes (effector cells; 100 .□L) isolated by Ficoll-Hypaque        separation (as described above) were then added to the target        cells to provide an effector/target cell (E:T) ratio of 10:1.        After 10 hours of incubation at 37° C., 20 .□l of a substrate        containing 5 .□g of        3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide        (MTT) was added. The mixture was incubated for 4 hours at 37° C.        after which the unmetabolized MTT was removed by aspiration. The        formazan crystals were dissolved by adding 200 □L of 95%        ethanol. Optical density was measured at 570 nm using a        microplate reader. The percentage of NK cell-specific lysis was        calculated as follows:        Specific Cytotoxicity (%)=100×{1−[(OD of target cells and        effector cells−OD of effector cells)/(OD of target cells)]}    -   3. Antibody response to vaccines: The test subjects are given an        array (up to 5) of vaccines after at least 12 weeks of probiotic        or control feeding. The vaccines may be a mixture of novel and        redundant vaccines. Non-limiting examples of vaccine arrays that        may be used include mixtures of vaccines prepared by Fort Dodge        Animal Health. Non-limiting examples of vaccines suitable for        use herein include Canine distemper, adenovirus, coronavirus,        parainfluenza, and parvovirus. The test subject's vaccine        history will determine the vaccines to be used. The specific        antibodies to the vaccines given are measured in blood for 3        weeks and the length and strength of response in control and        probiotic feeding groups compared.    -   4. Delayed-type hypersensitivity: An in vivo, non-invasive        method of assessing immune system status. This test comprises an        intradermal injection of the polyclonal mitogen        Phytohemmaglutinin (PHA) in combination with sheep red blood        cells a multivalent vaccine, histamine (100 μL of 0.0275 g/L        Histamine Phosphate; Greer, Lenoir, N.C.), or PBS (100 μL of        Phosphate Buffered Saline, 8.5 g/L; Sigma). The immune response        to the antigen is recorded as skinfold thickness using calipers        at time intervals of 0, 24, 48 and 72 hours post-injection. An        increase in skinfold thickness is indicative of a greater        hypersensitivity response that should be decreased by treatment        with the bacteria of the present invention.

Additional methods for determining the effect of the Bifidobacteriabacteria of the present invention are described in U.S. Pat. No.6,133,323 and U.S. Pat. No. 6,310,090.

Furthermore, ameliorating the effects of age may be determined usingdual x-ray absorptometry or CT scan for measuring body composition,including body fat mass, fat-free mass and bone mineral content.Similarly, this method may be used to determine anatomy changes such asweight loss or bone density in subjects following infection.

The Bifidobacteria of the present invention may also be used in a methodfor reducing stress levels in companion animals. Concentrations of bloodstress hormones including epinephrine, norepinephrine, dopamine,cortisol and C-reactive protein may be measured to determine stresslevels and their reduction or maintenance. These hormones are recognizedbiomarkers of stress and can be readily measured using techniques knownto those skilled in the art.

Further still, maintenance or improvement of the health of the skinand/or coat system of companion animals, including atopic disease of theskin, may be measured using skin and coat assessments conducted by twotrained individuals. Examples of criteria examined during suchassessments include:

-   -   a) Shedding index: A shedding index is assigned to each test        subject by collecting hair produced during a standardized        brushing session. The hair is retained and weighed, and control        and test subjects compared.    -   b) Subjective skin/coat evaluations: Trained panelists        subjectively evaluate skin and coat condition by assessing        shedding, dander, shine, uniformity, softness and density.    -   c) Skin functional assessment: The barrier function of the skin        may be assessed by wiping the skin surface with an        acetone-soaked gauze. This technique effectively disrupts the        skin barrier by removing single cell layers and associated lipid        fractions of the stratum corneum. Barrier disruption is        quantified by measuring the increase in transepidermal water        loss (TEWL) and the degree of redness of the insulted site using        methods known to those skilled in the art. Redness (erythema)        scores are obtained using the previously described camera and        lighting system. TEWL readings and redness scores are obtained        immediately before and after disruption, and at five and 24-hour        endpoints to assess the protective and healing properties of        skin.

The treatment or prevention of gastrointestinal infection, includingdiarrhoea, in companion animals may be measured using stool scores.Stools scores may be recorded daily according to the followingguidelines and control and test groups compared before and after feedingwith the bacteria according to the present invention.

Score: 5 Extremely Dry

This stool is hard and does not stick to surfaces. Stool will roll whenpushed. No indentations are made when stool is picked up. Stool is oftendefecated in groups of individual stools instead of one complete unit.The stool maintains original shape after collection.

Score: 4 Firm (Ideal Stool)

This stool is firm, well shaped, and cylindrical. This stool does notbreak apart easily when picked up. This stool may leave residue onsurfaces and gloves. This stool is often defecated as one unit. Thestool maintains original shape after collection.

Score: 3 Soft, With Shape

This stool is soft, however there are definite shapes. This stool willbreak apart easily and will definitely leave residue on surfaces andgloves. The stool often loses original shape after collection. Thisstool is often present with another score but can comprise whole stoolsample.

Score: 2 Soft, Without Shape

This stool is soft and will have no cylindrical shape. The shape oftenassociated with a “2” is a “cow patty” shape. This stool will lose theoriginal shape when collected and will definitely leave residue onsurfaces and gloves. This stool score is often present with anotherscore but can comprise the whole stool sample. This stool sample mayspread over an area of several inches.

Score: 1 Liquid

This stool score will always resemble liquid and there may or may not beparticulate matter present. This stool will often be defecated in groupsof piles instead of one complete unit. Mucous is often present with thisstool sample. This stool sample is very difficult to collect and residueis always left on surfaces and gloves. This stool sample may spread overan area of several inches.

In addition, other observations are also recorded, including: blood instool; foreign object in stool; or mucous in stool.

Furthermore, the treatment of gastrointestinal infection in companionanimals may comprise improving microbial ecology of companion animals.Improving the microbial ecology of companion animals preferablycomprises reducing the levels of pathogenic bacteria in the faeces ofcompanion animals. The levels of pathogenic bacteria present in thefaeces of companion animals may be enumerated using the standard platecount method known to those skilled in the art. More preferably, thepathogenic bacteria are selected from the group consisting ofClostridia, Escherichia, Salmonella, bacteriodes and mixtures thereof.Non-limiting examples of suitable strains of pathogenic bacteria includeC. perfringens, C. difficile, Eschericia coli, Salmonella typhimuriumand mixtures thereof.

The method of use of the bacteria of the present invention may alsoinclude the treatment, either prophylactic or therapeutic of the urinarytract of mammals, preferably companion animals. Non-limiting examples ofurinary tract treatment include treatment or prevention of urinary tractinfections, treatment or prevention of kidney disease, including kidneystones, treatment or prevention of bladder infections and the like.Without being bound by theory, it is believed that the Bifidobacteria ofthe present invention are useful in preventing these ailments as aresult of their ability to degrade oxalic acid, as demonstrated invitro. Oxalic acid is a by-product of urinary metabolism that can forminsoluble precipitates that result in kidney, bladder and other urinarytract infections. By degrading oxalic acid, and therefore potentiallypreventing its precipitation and build up in the urinary tract, thebacteria of the present invention may treat and prevent infections andother ailments of the urinary tract. Oxalic acid degradation may bemeasured in vitro using the Oxalic acid test kit cat #755699commercially available from Boehringer Mannheim/R-Biopharm.

The Bifidobacteria pseudolongum of the present invention may be used ina method for improving or maintaining the health of companion animalscomprising improving fiber digestion. Improving fiber digestion isdesirable as it promotes the growth of said probiotic bacteria, as wellas beneficial endogenous microflora, which aid in the suppression ofsome potentially pathogenic bacteria. In addition, a decrease in theamount of toxic metabolites and detrimental enzymes that result fromcolonic fermentation has been documented in humans (Tomomatsu, H.“Health effects of oligosaccharides”, (1994) Food Technol, 48, 61-65).Fiber digestion may be determined using the method described in Vickerset al. (2001), “Comparison of fermentation of selectedfructooligosaccharides and other fiber substrates by canine colonicmicroflora”, Am. J. Vet. Res. 61 (4), 609-615, with the exception thatinstead of inoculating using diluted fecal samples each experiment usedpure cultures of the bacterial strains of interest.

The method of use of the Bifidobacteria pseudolongum bacteria of thepresent invention typically involves oral consumption by the animal.Oral consumption may take place as part of the normal dietary intake, oras a supplement thereto. The oral consumption typically occurs at leastonce a month, preferably at least once a week, more preferably at leastonce per day. The Bifidobacteria pseudolongum bacteria of the presentinvention may be given to the companion animal in a therapeuticallyeffective amount to maintain or improve the health of the animal,preferably a companion animal. As used herein, the term “therapeuticallyeffective amount” with reference to the lactic acid bacteria, means thatamount of the bacteria sufficient to provide the desired effect orbenefit to a host animal in need of treatment, yet low enough to avoidadverse effects such as toxicity, irritation, or allergic response,commensurate with a reasonable benefit/risk ratio when used in themanner of the present invention. The specific “therapeutically effectiveamount” will vary with such factors as the particular condition beingtreated, the physical condition of the user, the duration of thetreatment, the nature of concurrent therapy (if any), the specificdosage form to be used, the carrier employed, the solubility of the doseform, and the particular dosing regimen.

Preferably, the lactic acid bacteria are given to the companion animalat a dose of from 1.0 E+04 to 1.0 E+14 CFU per day, more preferably from1.0 E+06 to 1.0 E+12 CFU per day. The composition preferably may containat least 0.001% of from 1.0 E+04 to 1.0 E+12 CFU/g of the Bifidobacteriapseudolongum obtainable by isolation from resected and washed canine GItract. The Bifidobacteria pseudolongum bacteria can be given to theanimal in either viable form, or as killed cells, or distillates,isolates or other fractions of the fermentation products of the lacticacid bacteria of the present invention, or any mixture thereof.

Preferably, the Bifidobacteria pseudolongum bacteria, or a purified orisolated fraction thereof, are used to prepare a composition intended tomaintain or improve the health of an animal. As indicated above, thecomposition may be part of the normal dietary intake, or a supplement.Where the composition comprises part of the normal dietary intake, thecomposition may be in the form of a dried animal food such as biscuitsor kibbles, a processed grain feed, a wet animal food, yogurts, gravies,chews, treats and the like.

Such compositions may comprise further components. Other components arebeneficial for inclusion in the compositions used herein, but areoptional for purposes of the invention. For example, food compositionsare preferably nutritionally balanced. In one embodiment, the foodcompositions may comprise, on a dry matter basis, from about 20% toabout 50% crude protein, preferably from about 22% to about 40% crudeprotein, by weight of the food composition. The crude protein materialmay comprise any material having a protein content of at least about 15%by weight, non-limiting examples of which include vegetable proteinssuch as soybean, cotton seed, and peanut, animal proteins such ascasein, albumin, and meat tissue. Non-limiting examples of meat tissueuseful herein include fresh meat, and dried or rendered meals such asfish meal, poultry meal, meat meal, bone meal and the like. Other typesof suitable crude protein sources include wheat gluten or corn gluten,and proteins extracted from microbial sources such as yeast.

Furthermore, the food compositions may comprise, on a dry matter basis,from about 5% to about 35% fat, preferably from about 10% to about 30%fat, by weight of the food composition. Further still, food compositionscomprising the lactic acid bacteria of the present invention may alsocomprise from about 4% to about 25% total dietary fiber. Thecompositions may also comprise a multiple starch source as described inWO99/51108.

The compositions of the present invention may further comprise a sourceof carbohydrate. Grains or cereals such as rice, corn, milo, sorghum,barley, alfalfa, wheat, and the like are illustrative sources. Inaddition, the compositions may also contain other materials such asdried whey and other dairy by products.

The compositions comprising the bacteria of the present invention mayalso comprise a prebiotic. “Prebiotic” includes substances or compoundsthat are fermented by the intestinal flora of the pet and hence promotethe growth or development of lactic acid bacteria in thegastro-intestinal tract of the pet at the expense of pathogenicbacteria. The result of this fermentation is a release of fatty acids,in particular short-chain fatty acids in the colon. This has the effectof reducing the pH value in the colon. Non-limiting examples of suitableprebiotics include oligosaccharides, such as inulin and its hydrolysisproducts commonly known as fructooligosaccharides,galacto-oligosaccarides, xylo-oligosaccharides or oligo derivatives ofstarch. The prebiotics may be provided in any suitable form. Forexample, the prebiotic may be provided in the form of plant materialwhich contains the fiber. Suitable plant materials include asparagus,artichokes, onions, wheat or chicory, or residues of these plantmaterials. Alternatively, the prebiotic fiber may be provided as aninulin extract, for example extracts from chicory are suitable. Suitableinulin extracts may be obtained from Orafti SA of Tirlemont 3300,Belgium under the trade mark “Raftiline”. For example, the inulin may beprovided in the form of Raftiline (g) ST which is a fine white powderwhich contains about 90 to about 94% by weight of inulin, up to about 4%by weight of glucose and fructose, and about 4 to 9% by weight ofsucrose. Alternatively, the fiber may be in the form of afructooligosaccharide such as obtained from Orafti SA of Tirlemont 3300,Belgium under the trade mark “Raftilose”. For example, the inulin may beprovided in the form of Raftilose (g) P95. Otherwise, thefructooligosaccharides may be obtained by hydrolyzing inulin, byenzymatic methods, or by using micro-organisms.

For dried pet foods a suitable process is extrusion cooking, althoughbaking and other suitable processes may be used. When extrusion cooked,the dried pet food is usually provided in the form of a kibble. If aprebiotic is used, the prebiotic may be admixed with the otheringredients of the dried pet food prior to processing. A suitableprocess is described in European patent application No 0850569. If aprobiotic micro-organism is used, the organism is best coated onto orfilled into the dried pet food. A suitable process is described inEuropean patent publication Number EP 0 862 863.

For wet foods, the processes described in U.S. Pat. Nos. 4,781,939 and5,132,137 may be used to produce simulated meat products. Otherprocedures for producing chunk type products may also be used; forexample cooking in a steam oven. Alternatively, loaf type products maybe produced by emulsifying a suitable meat material to produce a meatemulsion, adding a suitable gelling agent, and heating the meat emulsionprior to filling into cans or other containers. Typical wet foodcompositions may comprise from about 5% to about 15% protein, from about1% to about 10% fat, and from about 1% to about 7% fiber. Non-limitingingredients that may be used in wet food compositions include chicken,turkey, beef, whitefish, chicken broth, turkey broth, beef broth,chicken liver, brewers rice, corn grits, fish meal, egg, beet pulp,chloride, flax meal, lamb, beef by-products, chicken by-products andmixtures thereof.

In another embodiment, supplement compositions such as biscuits, chews,and other treats may comprise, on a dry matter basis, from about 20% toabout 60% protein, or from about 22% to about 40% protein, by weight ofthe supplement composition. As another example, the supplementcompositions may comprise, on a dry matter basis, from about 5% to about35% fat, or from about 10% to about 30% fat, by weight of the supplementcomposition. Food and supplement compositions intended for use bycanines or felines are commonly known in the art.

The pet foods may contain other active agents such as long chain fattyacids and zinc. Suitable long chain fatty acids include alpha-linoleicacid, gamma linolenic acid, linoleic acid, eicosapentanoic acid, anddocosahexanoic acid. Fish oils are a suitable source of eicosapentanoicacids and docosahexanoic acid.

Borage oil, blackcurrent seed oil and evening primrose oil are suitablesources of gamma linolenic acid. Safflower oils, sunflower oils, cornoils and soy bean oils are suitable sources of linoleic acid. These oilsmay also be used in the coating substrates referred to above. Zinc maybe provided in various suitable forms, for example as zinc sulfate orzinc oxide. Further, many ingredients commonly used in pet foods aresources of fatty acids and zinc. It has been observed that thecombination of chicory, as a source of prebiotic, with a linoleic-acidrich oil, such as soy bean oil, provides unexpected benefits, suggestiveof a synergistic effect.

Where the composition is in the form of a gravy, the compositionpreferably comprises at least 10% of a broth, or stock, non-limitingexamples of which include vegetable beef, chicken or ham stock. Typicalgravy compositions may comprise from about 0.5% to about 5% crudeprotein, from about 2% to about 5% crude fat, and from about 1% to about5% fiber.

Further non-limiting examples of supplements suitable for use hereininclude powders, oil suspensions, milk-based suspensions cheeses, andpills or capsules. Where the composition is in the form of a pill,suitable binding agents are required to maintain the pill in a solid,pressed form. Non-limiting examples of suitable binding agents includethe natural gums such as xanthan gum, pectins, lecithins, alginates andothers known to those skilled in the art. Where the composition is inthe form of a capsule, the composition is preferably encapsulated usingtechnologies known to those skilled in the art. Non-limiting examples ofsuitable encapsulation materials include polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), alginates, and gelatin. Yogurt-basedcompositions may comprise from about 1% to about 5% protein, from about10% to about 20% carbohydrate, from about 1% to about 5% fiber, fromabout 1% to about 5% fat and from about 50% to about 90% liquid carriersuch as milk.

EXAMPLES

The following examples are provided to illustrate the invention and arenot intended to limit the scope thereof in any manner.

Example 1 Isolation of Bifidobacteria Pseudolongum Bacteria From CanineGI Tracts

Canine intestinal samples were obtained from healthy dogs presenting atthe local veterinarians for owner initiated and approved euthanasia. Allanimals were healthy and disease-free. The colon, mid-colon, caecum andileum of each dog were dissected in order to expose the mucosa.

Supernatants were removed following agitation of the mucosal tissue(vortexed for 1 minute) and following mechanical homogenisation of thetissue. Each supernatant was plated on Reinforced Clostridia Agar (RCA)or MRS plus 0.05% cysteine plus mupirocin. These were incubatedanaerobically, using the Anerocult GasPak system, for 24 hours at 37° C.Isolated colonies from the plates were re-streaked onto either MRS orRCA and again grown anaerobically under the same conditions. Isolatedcolonies were re-streaked a further 4 times in order to purify a singlestrain. Colony morphology and microscopic appearance were assessed.Suitable isolates were tested for Gram reaction and catalase activity.Identification of gram positive, catalase negative rods was performedusing API testing (API 50CHL, BioMerieux). Harvested cells were washedtwice with 0.05M phosphate buffer (pH 6.5) and cysteine-HCl (500 mg/l)followed by sonication. Centrifugation removed cellular debris.Supernatants were incubated with NaF (6 mg/ml) and Na iodoacetate (10mg/ml) for 30 minutes at 37° C. The reaction was stopped by incubationwith hydroxylamine HCl (pH 6.5) for 10 minutes at room temperature.Colour development was monitored following the addition of HCl (4M),FeCl₃.6H₂O (5% (w/v) in 0.1M HCl) and fructose-6-phosphate (Na salt).Formation of acetyl phosphate from fructose-6-phosphate was evidenced bythe reddish colour formed by the ferric chelate of its hydroxymate.

Fifty-eight (58) lactic acid bacterial strains were isolated fromresected and washed canine GI tract, of which six were found to be ofthe genus Bifidobacteria, and one of the strain B. pseudolongum.

Example 2 Screening for Anti-Microbial Activity

The isolated Bifidobacteria pseudolongum bacterial strains wereincubated anaerobically in TPY broth. 2□1 of each culture were spottedonto TPY agar plates and incubated anaerobically overnight. Salmonellatyphimurium, Listeria monocytogenes, Listeria innocua and Eschericiacoli 0157H45 were pre-grown overnight and 100□1 inoculated into moltenagar (1% v/v). This indicator culture was poured onto the surface of theinoculated MRS or TPY plates. Following overnight incubation, zones ofinhibition around the probiotic colony were measured. All experimentswere performed in duplicate on three separate occasions. In addition,incorporating the buffer 2% betaglycerophosphate into the agar enabledassessment of the contribution of acid production to the observedpathogen inhibition in vitro.

The data presented in FIGS. 1, 2, 3 and 4 clearly demonstrate that theBifidobacteria pseudolongum bacteria strains of the present inventionobtainable by isolation from resected and washed canine GI tract haveanti-microbial activity in vitro, indicative of potential probioticactivity.

Example 3 In Vitro Measures of Survival And Colonisation PH Tolerance

Bacterial cells were harvested from overnight cultures, washed twice inphosphate buffer (pH 6.5) and resuspended in TPY broth adjusted with 1MHCl to pH 2.5. The cells were incubated anaerobically at 37° C. andtheir survival measured at intervals of 0, 30, 60, 120, 240 and 360minutes using the plate count method known to those skilled in the art.

FIG. 5 clearly demonstrates that nine strains were not pH 2.5 resistantover 1 hour, and the 49 strains were resistant to pH 2.5 over 1 hour.Table 2 summarises this data per strain.

TABLE 2 Strain Starting Conc. After 1 Viability designation Conc. hour(%) AHC A 1.50E+08 1.20E+08 80 AHC B 4.00E+07 5.50E+07 137 AHC C1.10E+08 1.50E+08 136 AHC F 6.00E+08 6.00E+08 100 AHC 7 2.50E+074.50E+07 180

Bile Resistance

The bacterial strains were streaked onto TPY agar supplemented withporcine bile (Sigma) at 0.5%, 1% and 5% (w/v). Plates were incubated at37° C. under anaerobic conditions and the growth recorded after 24hours. Growth was compared with control plates by an experiencedobserver, and the growth of colonies described as:

-   Negative (0)—no growth;-   +(1)—Hazy translucent growth (<33% control-plates with 0% bile);-   ++(2)—Definite growth but not as good as controls (>33% but <66%);-   +++(3)—Growth equivalent to controls (>66%).

Once the growth of the colonies in the presence of bile salts iscompared with the controls, the growth descriptors are given numericalvalues of 0, 1, 2 or 3 (−; +; ++, +++ respectively), and then expressedas a percentage, where 3 represents 100%.

FIG. 6 demonstrates that the Bifidobacteria of the present inventionclearly demonstrate a resistance to bile salts, being able to grow andform colonies at a level of at least 33% when exposed to 0.5% bilesalts.

Gut Epithelial Cell Adhesion

The human epithelial cell line, HT-29, was used to assess the adhesionproperties of selected strains. Epithelial cells were routinely culturedas a monolayer in 75 cm² tissue culture flasks at 37oC in a humidifiedatmosphere containing 5% CO₂ in Dulbecco's Minimal Essential Media(DMEM) containing 10% foetal calf serum (FCS), pen/strep, glutamine andfungizone. For experimental purposes, the epithelial cells were seededat a concentration of 5×10⁵ cells/ml (3 mls total volume) per well in 6well culture plates (Sarstedt). Following incubation for 7 days, toallow differentiation, the epithelial monolayers were washed withantibiotic-free medium containing 10% FCS. Bacterial suspensions plus/inantibiotic-free DMEM were added to each well and the cells incubated for90 minutes at 37° C. Following incubation, the monolayers were washedthree times with PBS. The epithelial cells were lysed in deionised H2Oand the number of adherent bacteria enumerated using the plate countmethod known to those skilled in the art. Adhesion was expressed as apercentage of the number of bacteria initially plated.

As can be seen from FIG. 7, the Bifidobacteria pseudolongum straindeposited with the NCIMB under deposition number NCIMB 41199 adhere toHT-29 gut epithelial cells at levels of at least 4%.

Example 4 16s-23s Intergenic Polynucleotide Sequencing

Bifidobacteria pseudolongum colonies were picked from an Agar plate andresuspended in IX PCR buffer, heated at 96° C. for 5 minutes, frozen at−70° C. for 5-10 minutes, thawed and an aliquot was added to a PCReppendorf tube. PCR was performed using the intergenic spacer (IGS)primers, IGS L: 5′-GCTGGATCACCTCCTTTC-3′ and IGS R:5′-CTGGTGCCAAGGCATCCA-3′. The cycling conditions were 96° C. for 1 min(1 cycle), 94° C. for 30 sec, 53° C. for 30 sec, 72° C. for 30 sec (28cycles). The PCR reaction contained 5 □l of DNA, PCR buffer (Bioline,UK), 0.2 mM dNTPs (Roche, UK), 0.4 □M IGS L and R primer (150 ng/50 □l)(MWG Biotech, Germany) and Bioline Taq polymerase (0.6 units). The PCRreactions were performed on a Hybaid thermocycler. The PCR products (8□l) were ran alongside a molecular weight marker (□X174 Hae III,Promega) on a 2% agarose EtBr stained gel in TAE, to determine their IGSprofile. Using the same primers as above, the intergenic spacer (IGS)DNA was sequenced for the 2 canine Bifidobacteria pseudolongum strainsusing methods known to those skilled in the art.

Following sequencing, the obtained sequences for the four depositedstrains were compared with the on-line sequence database “BLAST”,available at http://www.ncbi.nlm.nih.gov/BLAST/ for homology with otherdeposited bacterial 16s-23s sequences. Bifidobacterium pseudolongumATCC25865 was the closest match for AHC 7, having homology scores of92%. However, the several differences exist between these strains.

Example 5 Example Compositions

Examples 1 to 4 are examples of dried kibble compositions comprising theprobiotic Bifidobacteria pseudolongum of the present invention.

Percentage on a weight Basis Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4 Cerealgrains To 100 To 100 To 100 To 100 Poultry by-product meal 43.5 40 45 35Poultry fat 1.28 1.02 1.16 1.35 Egg product 2.4 2.1 2.5 2.2 Chickenliver meal 1.0 1.0 1.0 1.0 Brewer's dried yeast 1.0 1.0 1.0 1.0Monosodium phosphate 1.0 1.0 1.0 1.0 Calcium carbonate 0.8 0.8 0.8 0.8Potassium chloride 0.6 0.6 0.6 0.6 Vitamins 0.4 0.4 0.4 0.4 Cholinechloride 0.3 0.3 0.3 0.3 Minerals 0.3 0.3 0.3 0.3 DL-Methionine 0.1 0.10.1 0.1 Sodium Chloride 0.03 0.03 0.03 0.03 Probiotic (1 × 10¹⁰ cfu/g0.1 0.5 1 0.4 NCIMB 41199 in sunflower oil)

Examples 5 to 7 are examples of wet pet food compositions comprising theprobiotic Bifidobacteria pseudolongum of the present invention.

Percentage on a weight Basis Ingredient Ex. 5 Ex. 6 Ex. 7 Water To 38 To47 To 50 Poultry Liver To 25 To 20 To 15 Poultry Products 25 20 20Brewers Rice 5 7 10 Egg Product 3 2.5 1.5 Poultry Fat 2.9 3.0 3.2Chicken Stock 0.6 0.7 0.9 Taurine 0.1 0.1 0.1 Vitamins 0.05 0.1 0.1Minerals 0.05 0.1 0.1 Probiotic (1 × 10¹⁰ cfu/g 4 5 6 NCIMB 41199)

Examples 8 to 10 are examples of yogurt supplement compositionscomprising the probiotic Bifidobacteria pseudolongum of the presentinvention.

Percentage on a weight Basis Ingredient Ex. 8 Ex. 9 Ex. 10 Milk 82.7581.9 82.7 Sugar 12 12 10 Modified Starch 1.0 0.8 0.8 Prebiotic 0.25 0.30.5 Probiotic (1 × 10¹⁰ cfu/g 4 5 6 NCIMB 41199)

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed:
 1. A method of treating diarrhea in a canine comprisingorally administering to the canine a composition comprising an amount ofthe strain Bifidobacterium pseudolongum AHC7, NCIMB 41199, effective totreat diarrhea in the canine.
 2. The method of claim 1 wherein thecomposition is administered as part of a normal dietary intake.
 3. Themethod of claim 1 wherein the composition is administered as part of asupplement.
 4. The method of claim 1 wherein the composition isadministered at least once per month.
 5. The method of claim 1 whereinthe composition comprises is from about 10⁴ CFU to about 10¹² CFU of thestrain.
 6. The method of claim 1 wherein the strain is in viable form orin the form of killed cells.